Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold tremendous promise for both basic biology and cell based therapies due to their unlimited in vitro proliferation capacity and their potential to generate all tissue types (Murry and Keller 2008; Stadtfeld and Hochedlinger et al., 2010). Upon in vitro differentiation, these stem cell populations recapitulate early embryonic development, giving rise to a spectrum of mature cell types (Murry and Keller 2008).
During embryogenesis, the blastocyst inner cell mass gives rise to an epithelial population known as the epiblast. These cells traverse the primitive streak during gastrulation, giving rise to mesoderm and definitive endoderm (DE) (Lu et al., 2001). The epithelial sheet of nascent DE then folds to form the primitive gut tube consisting of three major domains along the anterior-posterior axis: the foregut, midgut and hindgut. These domains are further refined into specific regions from which the rudiments of various endodermal organs bud (Zorn and Wells, 2009). The foregut eventually gives rise to esophagus, trachea, lungs, thyroid, parathyroid, thymus, stomach, liver, biliary system and pancreas, while the midgut and hindgut form the small intestine and colon.
Endoderm-derived tissues, including liver and pancreas, are potentially useful for cell replacement therapies. It is possible to generate DE and its derivative lineages from PSCs in vitro through sequential exposure to cytokines that mimic embryonic morphogenesis. In this fashion, hepatic, intestinal and pancreatic cells can be produced from ESCs and iPSCs (D'Amour et al., 2006; Gouon-Evans et al., 2006; Basma et al., 2009; Spence et al., 2011). While these studies highlight the promise of PSC-derived endodermal tissues for transplantation therapies, several obstacles remain. Endodermal cells generated from PSCs tend to display immature phenotypes and in many instances are not fully functional. For example, most pancreatic β-cells currently generated in vitro from human ESCs are poly-hormonal and not glucose responsive (D'Amour et al., 2006; Nostro et al., 2011). In addition, the pluripotent nature of ESCs and iPSCs results in production of multiple cells types from different germ layers in most differentiation protocols. Thus, it is difficult to produce pure mono-lineage cultures of a desired cell type from PSCs (Murry and Keller, 2008). Finally, undifferentiated ESCs and iPSCs are tumorigenic and therefore, must be completely removed from their derivative tissues to be used for transplantation (Hentze et al., 2009).
It is an object of the present invention to provide culture and isolation methods that avoids these limitations.